KR20160145779A - Antibody Binding to FcRn for Treating Autoimmune Diseases - Google Patents

Abstract

The present invention relates to an isolated anti-FcRn antibody which is an antibody specific for FcRn (Neonatal Fc Receptor, abbreviation for fetal Fc receptor, FcRP, FcRB or Brambell receptor) which is a receptor having high affinity to IgG, A composition for treating an autoimmune disease comprising the antibody, and a method for treating and diagnosing an autoimmune disease using the antibody. The FcRn-specific antibody according to the present invention can be used for the treatment of auto-immune disease by non-recombinantly binding FcRn to IgG, thereby reducing the amount of anti-autoantibodies in blood.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a FcRn-specific human antibody and a composition for treating an autoimmune disease,

The present invention relates to an isolated anti-FcRn antibody which is an antibody specific for FcRn (Neonatal Fc Receptor, abbreviation for fetal Fc receptor, FcRP, FcRB or Brambell receptor) which is a receptor having high affinity to IgG, A composition for treating an autoimmune disease comprising the antibody, and a method for treating and diagnosing an autoimmune disease using the antibody. The FcRn specific antibody according to the present invention can be used for the treatment of autoimmune diseases by reducing the amount of auto-antibody in the blood by binding to FcRn in a non-competitive manner with IgG.

Antibodies are immunogenic proteins that bind to specific antigens. In most animals including humans and mice, heavy chain and light chain polypeptides are paired. Each chain has variable domains and constant And is composed of two distinct regions called constant domains. The variable regions of the heavy and light chains exhibit significant sequence diversity between the antibodies and are responsible for the specific binding characteristics to the target antigen and the constant regions have few inter-antibody sequence diversity and bind to a large number of natural proteins that induce important biochemical reactions I am responsible.

Under normal conditions, the half-life of most IgG except for IgG3 isotype in serum is about 22-23 days in humans, which is relatively longer than the serum half-life of other plasma proteins. Because of the long half-life of IgG, the IgG entering into the cell by pinocytosis strongly reacts to the neonatal Fc receptor (FcRn), a kind of Fc gamma receptor in the endosome at pH 6.0 , Which can evade the degradative lysosome pathway and, when circulated back to the cell membrane, IgG rapidly dissociates from FcRn in the blood stream at weakly basic pH (~ 7.4). By such receptor-mediated recycling mechanism, FcRn is known to effectively block IgG degradation in lysosomes and prolong the half-life of IgG (Roopenian et al., J. Immunol. 170: 3528, 2003) .

FcRn has also been shown to mediate the absorption of IgG antibodies from breast milk in rat neonatal intestines and to promote its transport into the circulatory system. FcRn has also been isolated in the human placenta, which is known to mediate the transport of maternal IgG into the fetal circulation and fetal circulation. In adults, FcRn is expressed in many tissues including the epithelium, nose, vagina, and biliary tree surface of the lung, bowel, and kidneys.

FcRn is typically a non-covalent heterodimer that resides within endosomes of endothelial cells and epithelial cells. FcRn is a membrane-bound receptor with three heavy chain alpha domains (α1, α2 and α3) and one soluble light chain β2-microglobulin (β2m) domain. Structurally, it belongs to a class of major histocompatibility complex (MHC) class 1 molecules that bear [beta] 2m as a common light chain. The αFcRn chain contains an ectodomain, a single-pass transmembrane, and a relatively short cytoplasmic tail containing a single oligosaccharide containing the three heavy chain domains α1, α2 and α3 and the β2m light chain domain tail) and has a molecular weight of about 46 kD.

To identify the role of FcRn on IgG homeostasis, when the genes encoding the? 2m and FcRn heavy chains of mice were "knocked out" and manipulated such that these proteins were not expressed, in these mice, the serum half-life of IgG And concentration, which suggests an FcRn-dependent mechanism for IgG homeostasis. It has also been suggested that anti-human FcRn antibodies can be produced in these FcRn knockout mice, and that these antibodies can prevent IgG binding to FcRn. The possibility of treating autoimmune disease by autoantibodies by decreasing IgG serum half-life due to inhibition of IgG binding to FcRn was revealed in a mouse model of autoimmune skin disease. (Li et al., J. Clin. Invest. 115: 3440, 2005). Therefore, an agent that blocks or antagonizes the binding of FcRn and IgG can be used in a method for treating or preventing IgG-mediated autoimmune diseases and inflammatory diseases.

An autoimmune disease is a disease caused by an immune system attacking its normal tissues, organs, or other body components, such as the nervous system, the gastrointestinal tract, the endocrine system, the skin, the skeletal system, It is a systemic disease that can occur in almost all parts of the body including blood vessels. It is known that autoimmune diseases occur in about 5% to 8% of the world population, but it is known that the prevalence is reported to be lower than the actual level due to limitations of understanding and diagnosis methods of autoimmune diseases.

The causes of autoimmune diseases have been studied for a long time in terms of genetic, environmental and immunological aspects, but the cause of definite disease has not yet been revealed. Recently, many studies have shown that many autoimmune diseases are caused by autoantibodies of IgG type. In fact, in the diagnosis and treatment of autoimmune diseases, the therapeutic effect of presence or absence of disease-specific autoantibodies Is widely known. Therefore, a large number of autoimmune diseases are known to have a disease-specific autoantibody and a pathological role thereof. If the autoantibody is removed from the blood, a rapid disease treatment effect can be obtained.

Autoimmune diseases and allogeneic diseases are diseases that are mediated by pathogenic antibodies such as immune neutropenia, Guillain-Barr syndrome, epilepsy, autoimmune encephalitis, Isaac's syndrome, syndrome, nevus syndrome, Pemphigus vulgaris, Pemphigus foliaceus, Bullous pemphigoid, epidermolysis bullosa acquisita, pemphigoid, pemphigus vulgaris, gestationis, mucous membrane pemphigoid, antiphospholipid syndrome, autoimmune anemia, autoimmune Grave's disease, Goodpasture's syndrome, myasthenia gravis, multiple myeloma, Multiple sclerosis, rheumatoid arthritis, lupus, and idiopathic thrombocytopenic purpu ra, ITP), lupus nephritis, and membranous nephropathy.

For example, in myasthenia gravis (MG), an autoantibody to acetylcholine receptor (AChR) located in the neuromuscular junction of the vas deferens muscle destroys or blocks the receptor, thereby inhibiting the contraction function of the vasculature It is known that when these autoantibodies are reduced, muscle function is restored.

In the case of idiopathic thrombocytopenic purpura (ITP), peripheral platelet destruction is caused by the development of an autoantibody that binds to a specific platelet membrane protein. Antiplatelet antibodies opsonize platelets and lead to rapid platelet destruction by reticular cells (eg, macrophages).

In general, attempts have been made to inhibit the immune system and increase the platelet concentration in order to treat ITP. The prevalence of ITP is higher in women than in men, and more prevalent in children than adults, with a prevalence of 1 per 10,000 population. Chronic ITP is one of the major blood disorders in both adults and children. In addition, it is causing a large amount of hospitalization and treatment costs in the US and worldwide. Twenty thousand new cases occur each year in the United States, and the costs associated with the treatment and special treatment of ITP are enormous. Most ITP pediatric patients have a very low number of platelets resulting in sudden bleeding (typical symptoms include left-sided, small red spots on the skin, nosebleeds, and gum bleeding). Pediatricians sometimes recover without treatment, but many doctors recommend careful observation and mitigation and intravenous injection of gamma globulin.

In the case of systemic lupus nephritis, lupus nephritis is a type of autoimmune disease in which immune complexes are accumulated in the organs of the whole body due to inadequate overproduction of autoantibodies such as antinuclear antibodies Inflammation is known to be an important cause of the reaction. Approximately 40% to 70% of these patients develop renal involvement, and about 30% of them develop into lupus nephritis. This is known as a malignant prognostic factor in the condition of Lupus, In the case of immunosuppressive therapy, no remission is reported, and even when the remission is reported, 10 to 65% of patients are reported to recur if the immunosuppressant is reduced. In conclusion, patients with severe Lupus nephritis (WHO class III and IV) die 5-10% after 10 years and 5-15% are serious diseases that lead to end-stage renal failure. .

Therefore, pathogenic antibody-induced autoimmune diseases such as the pemphigus pemphigus, neuromyelitis optica, and myasthenia gravis can be treated with antibodies against the novel mechanism of action to treat autoimmune diseases by eliminating the pathogenic autoantibodies (Pathogenic IgG-mediated autoimmune diseases ). Immune complex-mediated glomerular diseases such as lupus nephritis and membranous nephropathy are also expected to expand the therapeutic effect.

Methods of treating autoimmune diseases by intravenous drip (IVIG) of excess IgG in various autoimmune diseases have been widely used. (Arnson, Autoimmunity 42: 553, 2009). IVIG effects are explained by a variety of mechanisms, but one of them is also explained by mechanisms that increase the clearance of pathogenic antibodies by competition with endogenous IgG to FcRn. Intravenous administration of large amounts of human immunoglobulin (IVIG) has been shown to increase the platelet count of children suffering from immune ITP and to be beneficial for the treatment of multiple other autoimmune diseases. Many studies have identified mechanisms that work in the treatment of autoimmune diseases of IVIG. Regarding ITP, it has been reported that the effect of IVIG in early studies is mainly due to blocking Fc receptors involved in phagocytosis of antibody-opsonized platelets. Subsequent studies have shown that Fc-deficient IVIG leads to an increase in platelet count in some ITP patients. Recently, the effect of IVIG has been shown to stimulate the expression of FcγRIIb on macrophage cells, leading to inhibition of platelet phagocytosis There are also reports that it is caused by.

However, these IVIG treatments have significant side effects and are very expensive to administer. Other therapeutic methods used in the treatment of autoimmune / allogeneic immune diseases other than IVIG include a method using an immunosuppressive agent including polyclonal anti-D immunoglobulin, adrenocortical steroid, and chemotherapeutic agent, a cytokine plasma separation exchange method , Extracorporeal antibody adsorption (eg, using a Prosorba column), or surgical procedures such as splenectomy. However, as with IVIG, these therapies are also limited in their use due to insufficient efficacy and high cost. In addition, the competitive inhibition of the hypothetical mechanism by which IVIG inhibits FcRn binding results in a substantial increase in the clearance of pathogenic antibodies due to the low affinity for FcRn at physiological pH (i.e., pH 7.2-7.4) of IgG A fairly large amount of IVIG is required, with a typical clinical dose of IVIG of about 2 g / kg.

The use of inhibitors that have the concept of treating autoimmune diseases by competitively inhibiting the binding of IgG to FcRn is one of the promising therapies, but due to the high affinity of endogenous IgG for FcRn and the concentration of endogenous IgG in the blood It is known that a considerable amount of inhibitors are needed and therefore have the same limitations as current IVIG therapies.

Thus, although anti-FcRn antibodies are disclosed in WO2006 / 118772, WO2007 / 087289, WO2009 / 131702, and WO2012 / 167039, high affinity for FcRn can remove pathogenic antibodies in small quantities and reduce immunogenicity There is a need for development of improved human antibodies.

In order to solve the above problems, the present invention provides a therapeutic agent capable of efficiently and fundamentally treating an autoimmune disease including ITP, comprising an antibody capable of specifically binding to FcRn and a method for producing such an antibody And to provide the above objects. The FcRn-specific antibody according to the present invention is specific to FcRn and binds in a pH-independent manner to inhibit the binding of antibody Fc to FcRn non-competitively, resulting in an in vivo autoantibody The autoimmune disease can be treated.

It is a further object of the present invention to provide a method of treating a patient suffering from an autoimmune disorder selected from the group consisting of immunodeficiency neutropenia, gilanvalesindrome, epilepsy, autoimmune encephalitis, ischemic syndrome, idiopathic syndrome, imaginative pemphigus, decidual pemphigus, The present invention relates to the use of a compound of formula (I) for the manufacture of a medicament for the treatment or prevention of thrombocytopenic purpura, rheumatic arthritis, rheumatoid arthritis, idiopathic thrombocytopenia, autoimmune anemia, autoimmune Graft disease, (ITP), lupus nephritis, membranous nephropathy, and the like.

In order to accomplish the above object, the present invention provides a polypeptide having at least one amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: / RTI >

CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40 and SEQ ID NO: And

A CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: -FcRn antibody or fragment thereof.

The present invention also provides a pharmaceutical composition comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: CDR1 comprising an amino acid sequence representing homology;

An amino acid sequence having 90% or more homology with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: / RTI > And

An amino acid sequence having 90% or more homology with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: Lt; RTI ID = 0.0 > anti-FcRn < / RTI > antibody or fragment thereof.

The present invention also provides a pharmaceutical composition comprising a compound selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, FcRn antibodies or fragments thereof comprising at least one heavy chain and a light chain variable region comprising at least one amino acid sequence.

The present invention also provides a pharmaceutical composition comprising a compound selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, FcRn antibodies or fragments thereof comprising at least one heavy chain and light chain variable region comprising an amino acid sequence that is at least 90% identical to at least one amino acid sequence.

The present invention also provides polynucleotides encoding antibodies or fragments thereof.

The present invention also provides a kit for the treatment of a disease or condition selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, A polynucleotide encoding an anti-FcRn antibody or fragment thereof comprising one or more sequences is provided.

The present invention also provides a kit for the treatment of a disease or condition selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, A polynucleotide encoding an anti-FcRn antibody or fragment thereof comprising at least 90% homology with one or more sequences.

The present invention also provides a recombinant expression vector comprising the polynucleotide, a host cell transfected with the recombinant expression vector. The present invention additionally provides a method for producing an antibody, comprising culturing said host cell to produce an antibody; And

Separating and purifying the resulting antibody to recover an antibody that specifically binds to FcRn, or a method for producing the fragment.

The present invention also provides a pharmaceutical composition comprising said antibody or fragment thereof, and at least one pharmaceutically acceptable carrier.

The present invention also provides a method of treating an autoimmune disease patient comprising administering the composition to a patient.

The present invention also provides a composition comprising said antibody or fragment thereof, and a detection label.

The present invention further provides a method of detecting FcRn in vitro or in vivo, including using the antibody or fragment thereof.

The antibody or fragment thereof according to the present invention specific for FcRn, which is a receptor having high affinity for IgG, has high affinity and specificity, has little problem due to immunogenicity, and binds FcRn with IgG, etc. in a non- Since it has the property of reducing the amount of antibody in blood, it can be very useful for the treatment and diagnosis of autoimmune diseases.

FIG. 1 is a graph showing the results of analyzing HL161A, HL161B, HL161C and HL161D antibody proteins obtained through antibody expression using CHO-S cells and protein A purification on a SDS-PAGE gel in a reduced or non-reduced state, HL161 antibodies were found to form a complete human IgG1 type structure of about 160 kDa in the non-reducing state, and it was confirmed that in the reduced state, the heavy chain was about 55 kDa and the light chain was about 25 kDa, which was a typical antibody structural unit. 1, Lane 1 represents an MW marker, Lane 2 represents a non-reduced state (* NEM-treated) of 2 μg, and Lane 3 represents a reduced state of 2 μg.
Figure 2 shows the results of analysis using SPR instrument to determine the kinetic analysis (KD) of four anti-FcRn antibodies, HL161A, HL161B, HL161C and HL161D binding to FcRn. Proteon GLC chip The interaction of human FcRn with HL161A, HL161B, HL161C or HL161D antibodies was analyzed at pH 6.0 and pH 7.4 using Proteon XPR36 (Bio-Rad)
Figure 2a shows the results of the interaction between human FcRn and HL161A antibody at pH 6.0.
Figure 2b shows the results of the interaction of human FcRn with HL161A antibody at pH 7.4.
FIG. 2C shows the result of analyzing the interaction between human FcRn and HL161B antibody at pH 6.0. FIG.
FIG. 2d shows the result of analyzing the interaction between human FcRn and HL161B antibody at pH 7.4. FIG.
Figure 2E is the result of analyzing the interaction of human FcRn with HL161C antibody at pH 6.0.
FIG. 2f shows the result of analyzing the interaction between human FcRn and HL161C antibody at pH 7.4.
Figure 2g shows the result of analyzing the interaction of human FcRn with HL161D antibody at pH 6.0.
Figure 2h shows the result of analyzing the interaction of human FcRn with HL161D antibody at pH 7.4.
FIG. 3 is a graph showing the binding ability of the selected two kinds of antibodies to human FcRn (hFcRn) on the cell surface. HL161A and HL161B antibodies bound to human FcRn present on the cell surface were incubated in HEK293 cells overexpressing human FcRn And the resultant antibody was confirmed to bind to FcRn on the cell surface at pH 6.0 and pH 7.4. Human FcRn binding of the HL161A and HL161B antibodies was performed by treating each antibody with a pH-dependent cell and then using a Alexa488 labeled anti-human goat antibody in a Fluorescent activated cell sorter FACS) analysis.
FIG. 4 is a graph showing the results of analysis of inhibitory effect on human IgG and human FcRn expressing cells at pH 6.0, showing whether the two selected antibodies binding to the cell surface human FcRn can inhibit the binding of human IgG to human FcRn And Fig. HL161A and HL161B antibodies confirmed to bind to human FcRn overexpressing HEK293 cells were sequentially diluted four times from 200 nM to obtain a profile of binding inhibition of Alexa488 labeled human IgG to human FcRn.
FIGS. 5A and 5B are graphs showing the effect of the antibodies HL161A and HL161B selected from Tg32 (hFcRn + / +, hβ2m + / +, mFcRn - / -, mβ2m - / -) transfected with human FcRn in higG1 The results are as follows. At 0 hour, 5 mg / kg of biotin-hIgG and 495 mg / kg of human IgG were administered intraperitoneally to saturate IgG in the body. The administration of the drug was carried out by intraperitoneal (ip) administration of human IgG1, HL161A, HL161B or PBS once a day at a dose of 5, 10 and 20 mg / kg at 24, 48, 72 and 96 hours after administration of biotin-IgG Results. Sample collection was performed 24, 48, 72, 96, 120, 168 hours after biotin-IgG administration. At 24, 48, 72, and 96 hours, blood samples were taken before drug administration and the amount of biotin- Respectively. The results are expressed as the relative amounts of the remaining amounts at each time point, with the remaining amount of the 24-hour blood sampling sample as 100%.
FIG. 6 is a graph showing the results of analysis of changes in blood of monkey IgG by administration of two antibodies to HL161A and HL161B using a cynomorphic monkey having 96% sequence homology with human FcRn, wherein HL161A and HL161B antibodies As a result of intravenous administration of monkeys at a dose of 5, 20 mg / kg to monkeys monkeys, monkey IgG decreased up to 70% compared to 0 hours and decreased 30% until 29 days.
FIG. 6A shows the IgG reduction effect in monkey blood by HL161A and HL161B antibody concentrations.
6B shows the effect of HL161A and HL161B antibody (5 mg / kg concentration) on blood IgG reduction by monkey species.
Fig. 6C shows the effect of HL161A and HL161B antibody (20 mg / kg concentration) on blood IgG reduction by monkey species.
7A and 7B are graphs showing the results of analysis of the pharmacokinetic profiles of HL161A and HL161B in a test using Cynomolgus monkeys. As a whole, HL161B exhibited a higher half-life, AUC and Cmax than HL161A.
8A to 8C are diagrams showing the results of analysis of changes in blood of monkey IgM, IgA, and albumin due to the administration of HL161A and HL161B antibodies in a test using Cynomolgus monkey, It was judged to be a result of individual differences rather than the effect of the test substance because it was a change within the normal range of the Cynomolgus monkey,
Figure 8a shows the change in monkey IgM in monkey blood.
8B shows the change in the monkey IgA in the monkey blood.
Figure 8c shows monkey albumin changes in monkey blood.

In order to achieve the above object, the present invention provides an antibody having a high affinity to FcRn and capable of binding in a specific, pH-independent manner and consisting of a sequence derived from a human, do.

The antibody provided in the present invention is a binding molecule having specificity for FcRn. The antibody may be a monoclonal antibody (e.g., a full length antibody having an immunoglobulin Fc region), an antibody composition with multiple epitope specificities, a multispecific antibody (e. G., A bispecific antibody), a diabody But are not limited to, single chain molecules as well as antibody fragments (e.g., Fab, F (ab ') 2, and Fv). The antibody provided herein may be, for example, a monoclonal antibody to human FcRn.

The monoclonal antibody may comprise a mouse antibody. In addition, monoclonal antibodies may be obtained by immunizing a mammal with a heavy chain and / or light chain wherein some of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species, e. G. Mouse or belonging to a particular antibody class or subclass, Quot; chimeric "antibodies that are identical or homologous to corresponding sequences in antibodies derived from or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity. "Humanized antibodies" are used as a subset of "chimeric antibodies ".

As an alternative to humanization, human antibodies can be generated. A "human antibody" is an antibody produced by a human or having an amino acid sequence corresponding to an antibody produced using any human antibody production technique. Human antibodies can be prepared using a variety of techniques known in the art, including phage display libraries. Human antibodies can be made by administering an antigen to a transgenic animal that has been modified to produce antibodies in response to antigen challenge inoculation but whose endogenous locus is not functioning, such as an immunized xenomouse . The antibodies provided herein may, for example, have the form of human antibodies.

The basic four-chain antibody unit is a heterodimeric tetravalent protein consisting of two identical light chains (L) and two identical heavy chains (H). The light chain has a variable region (VL) at its N-terminus followed by a constant region at its other end. The heavy chain has a variable region (VH) at the N-terminus followed by three constant regions (CH) for the? And? Chains, and four constant regions (CH) for the? And? Isomorphs, respectively.

'Variable' indicates that a particular region of the variable region is significantly different in sequence between antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. Variability is concentrated in three segments called the hypervariable region (HVR), or CDR, in both the light and heavy chain variable regions. The more highly conserved portion of the variable region is called the framework region (FR). The heavy and light chain variable regions have FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 structures from the N-terminus to the C-terminus.

In the present invention, an antibody having affinity and specificity to human FcRn was obtained using a human immunoglobulin transgenic animal. Transgenic animals can be produced by inactivating the germ line gene of an animal and transplanting a human Ig germline gene locus. In the case of using a transgenic animal, the antibody is naturally optimized by the immune system of the animal, and the affinity maturation is not needed, so that it is possible to develop an antibody drug having a low affinity and high affinity in a short period of time US20090098134, US20100212035, Menoret et al, Eur J Immunol, 40: 2932, 2010).

In the present invention, OmniRat ( ^TM ) of OMT (USA), which has patented technology for human immunoglobulin transgenic mice, was utilized. OmniRat ^TM is a vector comprising a heavy chain consisting of a CH2, CH3 domain and a V, D, J domain and a CH1 domain consisting of a human gene and comprising human kappa light chain and lambda light chain efficiently, human FcRn (Menoret et al., Eur J Immunol, 40: 2932, 2010).

In order to obtain high affinity monoclonal antibody against FcRn, human FcRn was injected into a transgenic mouse (OmniRat ( ^TM )), and then B cells were extracted and fused with myeloma cells to prepare a hybridoma The antibody produced from the hybridoma was purified.

The antibody according to the present invention acts as a non-competitive inhibitor of IgG in binding to FcRn. Binding of the pathogenic antibody to FcRn is inhibited by the FcRn binding with the antibody according to the present invention, and thus the clearance of the pathogenic antibody from the body of the pathogenic antibody is promoted from the body of the individual, thereby reducing the half-life.

As used herein, the term "pathogenic antibody" refers to an antibody that causes a pathological condition or disease. Such an antibody may be, for example, an anti-platelet antibody, an anti-acetylcholine antibody, an anti-phospholipid antibody, an anti-collagen antibody, an anti-ganglioside antibody, Anti-desmoglein antibody, and the like, but are not limited thereto.

The antibodies according to the present invention have the advantage of being capable of non-competitively inhibiting the binding of the pathogenic antibody with FcRn at physiological pH, i.e. pH 7.0-7.4. FcRn binds its ligand, i. E., IgG, in a pH-dependent manner, and does not exhibit substantial affinity for IgG at non-acidic physiological pH. Thus, an anti-FcRn antibody that specifically binds FcRn at physiological pH acts as a non-competitive inhibitor to the binding of IgG to FcRn, where the binding of the anti-FcRn antibody to FcRn affects the presence of IgG I do not accept. Thus, an antibody according to the present invention that binds FcRn non-competitively to IgG by pH-independence can inhibit the FcRn mediated binding of IgG to a competitive inhibitor, i. E., At a much lower concentration than conventional antibodies that competitively bind FcRn with IgG It is possible to treat diseases caused by signal transduction. In addition, the anti-FcRn antibody according to the present invention maintains binding with higher affinity than serum IgG to FcRn in the process of intracellular movement in the state of binding with FcRn. Therefore, It is possible to inhibit the binding of IgG and FcRn even in the culture medium, and it is possible to promote the clearance of IgG.

The antibody according to the present invention has an affinity for FcRn even at physiological pH, that is, an environment in which IgG does not bind to FcRn, that is, a pH of 7.0 to 7.4, and has a higher affinity for FcRn than IgG in blood at pH 6.0, Lt; / RTI >

In one aspect, the invention provides a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: CDR1;

CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40 and SEQ ID NO: And

A FcRn comprising CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: Antibodies or fragments thereof.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention, ≪ / RTI >

In addition, sequences having homology with the nucleotide sequence and the amino acid sequence described in SEQ ID NO: 2 are also included in the scope of the present invention so long as the object of the present invention can be achieved. "Homology" refers to a degree of similarity to one or more nucleotide or amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 44, comprising 90% or more identical sequences, preferably 95% or more, 96% or more, 97 , At least 98%, at least 99% identical to the sequence. Comparison of homology is performed using a known visual or comparison program. Commercially available computer programs can calculate homology between two or more sequences as a percentage (%). The homology (%) can be calculated for an adjacent sequence.

Furthermore, as long as the object of the present invention can be achieved, an antibody specifically binding to FcRn having a KD (dissociation constant) of 0.01 to 2 nM under the conditions of pH 6.0 and pH 7.4 may be included in the scope of the present invention. As used herein, "KD" means an equilibrium dissociation constant for binding of an antibody antigen, KD = kd / ka (ka means association rate constant and kd means dissociation rate constant ), And the measurement of kd or ka can be carried out at 25 ° C or 37 ° C.

In one embodiment, the antibody or fragment thereof of the invention comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23,

CDR1 comprising the amino acid sequence of SEQ ID NO: 27, CDR2 comprising the amino acid sequence of SEQ ID NO: 28 and CDR3 comprising the amino acid sequence of SEQ ID NO: 29,

CDR1 comprising the amino acid sequence of SEQ ID NO: 33, CDR2 comprising the amino acid sequence of SEQ ID NO: 34 and CDR3 comprising the amino acid sequence of SEQ ID NO: 35, or

CDR1 comprising the amino acid sequence of SEQ ID NO: 39, CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR3 comprising the amino acid sequence of SEQ ID NO: 41.

The amino acid sequence represented by the sequence number may be an amino acid sequence corresponding to CDR1 to CDR3 of the heavy chain variable region.

In another embodiment, the antibody or fragment thereof of the invention comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a CDR2 comprising the amino acid sequence of SEQ ID NO: 25 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 26,

CDR1 comprising the amino acid sequence of SEQ ID NO: 30, CDR2 comprising the amino acid sequence of SEQ ID NO: 31 and CDR3 comprising the amino acid sequence of SEQ ID NO: 32,

CDR1 comprising the amino acid sequence of SEQ ID NO: 36, CDR2 comprising the amino acid sequence of SEQ ID NO: 37 and CDR3 comprising the amino acid sequence of SEQ ID NO: 38, or

A CDR1 comprising the amino acid sequence of SEQ ID NO: 42, a CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and an amino acid sequence of SEQ ID NO: 44.

The amino acid sequence represented by the sequence number may be an amino acid sequence corresponding to CDR1 to CDR3 of the light chain variable region.

The antibody or fragment thereof according to the present invention specifically comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: And a light chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 24, CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR3 comprising the amino acid sequence of SEQ ID NO: 26,

A heavy chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 27, CDR2 comprising the amino acid sequence of SEQ ID NO: 28 and CDR3 comprising the amino acid sequence of SEQ ID NO: 29, and CDR1 comprising the amino acid sequence of SEQ ID NO: A light chain variable region comprising CDR2 comprising the amino acid sequence of SEQ ID NO: 31 and CDR3 comprising the amino acid sequence of SEQ ID NO: 32,

CDR1 comprising the amino acid sequence of SEQ ID NO: 33, CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR3 comprising the amino acid sequence of SEQ ID NO: 35 and CDR1 comprising the amino acid sequence of SEQ ID NO: A light chain variable region comprising CDR2 comprising the amino acid sequence of SEQ ID NO: 37 and CDR3 comprising the amino acid sequence of SEQ ID NO: 38, and

A heavy chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 39, CDR2 comprising the amino acid sequence of SEQ ID NO: 40 and CDR3 comprising the amino acid sequence of SEQ ID NO: 41, and CDR1 comprising the amino acid sequence of SEQ ID NO: A light chain variable region comprising CDR2 comprising the amino acid sequence of SEQ ID NO: 43 and CDR3 comprising the amino acid sequence of SEQ ID NO: 44, and a light chain variable region comprising the light chain variable region comprising the amino acid sequence of SEQ ID NO: 44.

In one embodiment, an antibody or fragment thereof according to the present invention comprises an antibody or fragment thereof selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, And at least one heavy chain and light chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 20.

Specifically, the antibody or fragment thereof according to the present invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 and / , SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

The antibody or fragment thereof according to the present invention specifically comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12,

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14,

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16,

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18, and

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20, and a light chain variable region.

&Quot; Fragment "or" antibody fragment "according to the present invention refers to a polypeptide that does not comprise a full length antibody polypeptide but is derived from an antibody polypeptide molecule (e.g., antibody heavy chain or light chain polypeptide) And at least a portion of the full-length antibody polypeptide. Antibody fragments are not limited to cleavage fragments, but often include polypeptides that comprise cleavage sites of full length antibody polypeptides. As the term is used with reference to an antibody, the fragment may not bind to the antigen by itself, because the fragment includes a fragment comprising an antibody polypeptide-derived single chain polypeptide (e.g., heavy chain or light chain antibody polypeptide).

Antibodies according to the invention also include fragments of the antibody. Fragments of the antibody in the present invention may be any of the following: multispecific antibodies such as single chain antibodies, bispecific antibodies, triple specific antibodies, diabodies, triabodies, tetrabodies, Fab fragments, F (ab ') ₂ fragments, Fd, scFv, , Dual-specific antibodies, minibodies, sterol regulatory binding protein cleavage activating proteins, chelating recombinant antibodies, tri-bodies, bibodies, intrabodies, nanobodies, small modular immunopharmaceuticals A protein having binding ability to FcRn, a domain immunoglobulin fusion protein, a camelized antibody, an antibody containing VHH, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, But are not limited to, synthetic antibodies based on scaffolds.

It will be apparent to those of ordinary skill in the art that as long as the binding function to FcRn is maintained, fragments of any type of antibody according to the present invention will exhibit the same characteristics as the antibodies according to the present invention.

Also, as long as the characteristics of the antibody or fragment of the present invention are maintained, the antibody in which the mutation occurs in the variable region is also included in the scope of the present invention. An example of such an antibody is conservative substitution of amino acids in the variable region. Conservative substitutions refer to substitution with other amino acid residues that have similar properties to the original amino acid sequence, for example, lysine, arginine, and histidine have similar characteristics with their base side chains, and aspartic acid and glutamic acid They have similar characteristics in that they have acid side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine and tryptophan have similar characteristics in that they have non-negatively polarized side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, In terms of having side chains, tyrosine, phenylalanine, tryptophan, and histidine have similar characteristics in that they have aromatic side chains. Therefore, it is obvious to those of ordinary skill in the art that even when amino acid substitution occurs within the group having similar characteristics as described above, there will be no change in characteristics. Therefore, as long as the characteristics of the antibody according to the present invention are maintained, The present invention also includes an antibody having a mutation caused by conservative substitution in the present invention.

The antibody or fragment thereof according to the present invention may also be used in the form of a conjugate conjugated with another substance. Examples of the substance that can be conjugated to the antibody or the fragment thereof according to the present invention include therapeutic agents used for the treatment of autoimmune diseases and substances capable of inhibiting the activity of FcRn or substances of blood, Is physically connected to a moiety that improves its stabilization and / or maintenance within the circulatory system. For example, an FcRn-binding antibody may be conjugated to a polymer, for example, a non-antigenic polymer such as a polyalkylene oxide or a polyethylene oxide. Suitable polymers will vary substantially by weight. Polymers having an average molecular weight in the range of about 200 to about 35,000 (or about 1,000 to about 15,000, and 2,000 to about 12,500) may be used. For example, an FcRn-binding antibody may be conjugated to a water soluble polymer, for example, a hydrophilic polyvinyl polymer, such as polyvinyl alcohol and polyvinylpyrrolidone. Examples of such polymers include, but are not limited to, homopolymers, copolymers, and block copolymers of polyalkylene oxides such as polyethylene glycol or polypropylene glycol, polyoxyethylene polyol, and the like. As a clue, the water solubility of these block copolymers should be maintained.

In another aspect, the present invention relates to a pharmaceutical composition comprising the antibody or fragment thereof, and at least one pharmaceutically acceptable carrier, and a pharmaceutical composition for treating an autoimmune disease. The present invention also relates to a method of treating an autoimmune disease comprising administering to a patient in need of such treatment an effective amount of an antibody or fragment thereof that specifically binds to FcRn.

The pharmaceutical composition may additionally contain conventional pharmaceutically acceptable carriers and excipients. The pharmaceutically acceptable carrier should be compatible with the active ingredient of the antibody or the like according to the present invention and may be any one of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, Two or more components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, a diluent, a dispersant, a surfactant, a binder, and a lubricant may be additionally added to prepare a formulation for injection, such as an aqueous solution, a suspension or an emulsion. It may also be formulated into various forms such as powders, tablets, capsules, liquids, injections, ointments, syrups and the like and may be formulated in unit-dose or multi-dose containers such as sealed ampoules and bottles May be provided.

The pharmaceutical composition may be applied to all autoimmune diseases mediated by IgG and FcRn. Typical diseases include immunodeficiency neutropenia, gilanvalesindrome, epilepsy, autoimmune encephalitis, ischemic syndrome, birth syndrome, A myopathic syndrome, autoimmune anemia, autoimmune gravida, Gupta stewer syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, Arthritis, lupus, and idiopathic thrombocytopenic purpura (ITP), lupus nephritis, membranous nephropathy, and the like.

The dose of the antibody in the treatment method according to the present invention can be suitably determined in consideration of the severity, condition, age, disease history, etc. of the patient and can be administered, for example, in a dose of 1 mg / kg to 2 g / kg. The antibody may be administered singly or several times.

The invention provides a method of alleviating an autoimmune or allogeneic immune condition comprising administering an antibody or fragment thereof according to the invention to a subject in need of treatment, while at the same time providing certain anti-FcRn therapies.

The method for alleviating the autoimmune or allogeneic immune status according to the present invention or the anti-FcRn therapy can be achieved by administering the pharmaceutical composition according to the present invention to a subject, wherein the pharmaceutical composition according to the present invention can be administered orally or parenterally For example, oral, intravenous, intramuscular, intraarterial, intramedullary, intradermal, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, sublingual or topical administration, no. The dosage of the pharmaceutical composition according to the present invention may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, method, excretion rate and severity of disease, Can be determined easily, and can be administered once or repeatedly. Generally 1 to 200 mg / kg, more preferably 1 to 40 mg / kg, can be administered to patients with autoimmune or allogeneic immune symptoms, and this dosage regimen is preferably that the serum intrinsic IgG concentration is less than 75% And can be applied intermittently or chronicly (continuous) in view of the patient's condition.

The present invention also provides a diagnostic composition comprising the antibody or fragment thereof according to the present invention and a diagnostic method using the same. That is, the antibody or fragment thereof binding to FcRn according to the present invention has in vitro and in vivo diagnostic utility.

In another aspect, the present invention relates to a composition for detecting FcRn comprising an antibody or fragment thereof that specifically binds to FcRn. The present invention also relates to a method for detecting the presence of FcRn in vitro or in vivo, comprising the step of treating an antibody or fragment thereof that specifically binds to said FcRn.

In vitro detection methods include, for example, (1) contacting a sample with an FcRn-binding antibody; (2) detecting the formation of a complex between the FcRn-binding antibody and the sample; And / or (3) contacting a reference sample (e.g., a control sample) with the antibody; (4) determining the degree of formation of the complex between the antibody and the sample as compared to that in the reference sample. A change in the formation of a complex in a sample or an individual as compared to a control sample or an individual, for example, a statistically significant change, may mean the presence of FcRn in the sample.

In vivo detection methods include (1) administering an FcRn-binding antibody to an individual; (2) detecting the formation of a complex between the FcRn-binding antibody and the individual. Detection may include determining the location or point of time of complex formation. An FcRn-binding antibody can be directly or indirectly labeled with a detectable substance that facilitates detection of the bound or unbound antibody. Suitable detectable materials include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Complex formation between FcRn-binding antibodies and FcRn can be detected by measuring or visualizing antibodies bound to FcRn or antibodies that are not bound. Conventional detection assays, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry, may be used. In addition to the labeling of FcRn-binding antibodies, the presence of FcRn can be analyzed in samples by competitive immunoassay using labeled reference and non-labeled FcRn-binding antibodies. In one example of this assay, the amount of labeled reference bound to a biological sample, a labeled reference, and an FcRn-binding antibody and bound to an unlabeled antibody is measured. The amount of FcRn in the sample is inversely proportional to the amount of labeled reference bound to the FcRn-binding antibody.

For use for diagnostic purposes, an antibody or fragment thereof according to the present invention may be labeled with a fluorophore or chromophore. Since antibodies and other proteins absorb light having wavelengths up to approximately 310 nm, the fluorescence moiety should be selected to have substantial absorption at wavelengths greater than 310 nm, preferably greater than 400 nm. A variety of suitable fluorescent materials and chromophores can be labeled with a fluorescent chromophore group. Among the fluorescent chromophore groups, a group of fluorescent materials are xanthene dyes, including fluoresceins and rhodamines. Another group of fluorescent compounds is naphthylamine. Once labeled with a fluorescent moiety or chromophore, the antibody detects the presence or localization of FcRn in the sample, for example, using fluorescence microscopy (e.g., confocal or deconvolution) Can be used.

The presence or localization of FcRn using the antibody or fragment thereof according to the present invention can be detected by various methods such as histological analysis, protein arrays, FACS (fluorescence activated cell sorting), and the like.

In the present invention, the presence of an in vivo FcRn or FcRn-expressing tissue can be confirmed by an in vivo image detection method. (1) administering to an individual (e.g., an autoimmune disease patient) an anti-FcRn antibody conjugated to a detectable marker; (ii) exposing said subject to a means for detecting a detectable marker for an FcRn-expressing tissue or cell. For example, the subject may be imaged by, e.g., NMR or other tomography. Examples of labels useful for diagnostic imaging include radioactive labels, fluorescent labels, positron emitting isotopes, chemiluminescent materials, luminescent enzymes, and the like. The radiolabeled antibody can also be used for in vitro diagnostic tests. The specific activity of an isotope-labeled antibody depends on half life, isotopic purity of the radioactive label, and how the label is incorporated into the antibody.

The present invention also relates to antibodies or fragments thereof that bind to FcRn and to methods for their diagnostic use, for example, in vitro, for example in a sample, for example in a biopsy or in a cell from an autoimmune disease patient, Within the scope of the invention, a kit comprising instructions for use of an FcRn-binding antibody or antigen-binding fragment thereof for detecting FcRn, for example, by imaging an individual. The kit may further comprise at least one additional reagent, for example a label or an additional diagnostic agent. In the case of in vivo use, the antibody may be formulated as a pharmaceutical composition.

In another aspect, the present invention relates to a polynucleotide encoding an antibody or fragment thereof that specifically binds to said FcRn.

In one embodiment, the antibody is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: FcRn < / RTI > antibodies or fragments thereof that exhibit at least 90% homology with one or more of the sequences of SEQ ID < RTI ID = 0.0 >

Specifically, the polynucleotide sequence encoding the heavy chain of an antibody according to the present invention comprises a polynucleotide sequence encoding a light chain of an antibody according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 and / The polynucleotide sequence is SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO:

In another aspect, the present invention provides a recombinant expression vector comprising the polynucleotide, a host cell transfected with the recombinant expression vector, and an antibody or fragment thereof that specifically binds FcRn using the recombinant expression vector and the host cell And a method for manufacturing the same.

In one embodiment, the antibody or fragment thereof is preferably produced by expressing and purifying by a recombinant method. Specifically, the variable region encoding the antibody specifically binding to FcRn according to the present invention is separately prepared, Or simultaneously expressed in one host cell.

As used herein, the term "recombinant vector" refers to an expression vector capable of expressing a target protein in a suitable host cell, and a gene construct comprising an essential regulatory element operably linked to the expression of the gene insert. In the present invention, "operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein to perform a general function. The operative linkage with a recombinant vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be carried out by using enzymes generally known in the art to which the present invention belongs Can be easily carried out using

Suitable expression vectors that may be used in the present invention may include signal sequence for membrane targeting or secretion in addition to expression control elements such as promoter, initiation codon, termination codon, polyadenylation signal and enhancer. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and must be operative in the subject when the gene construct is administered and in the coding sequence and in frame. Generic promoters may be constitutive or inducible. Prokaryotic cells include, but are not limited to, the lac, tac, T3 and T7 promoters. Eukaryotic cells include monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) such as long terminal repeat (LTR) promoter of HIV, moloney virus promoter, Human β-actin promoter, human hepatoblast, human muscle creatine (CMV) promoter, epstein barr virus (EBV) promoter, rous sarcoma virus (RSV) promoter as well as the cytomegalovirus , And promoters derived from human metallothionein, but are not limited thereto. The expression vector may comprise a selection marker for selecting a host cell containing the vector. Selection markers are for selecting cells transfected with a vector, and markers conferring selectable phenotypes such as drug resistance, nutritional requirement, resistance to cytotoxic agents or expression of surface proteins may be used. Since only the cells expressing the selectable marker survive in the selective agent treated environment, the transfected cells are selectable. In addition, if the vector is a replicable expression vector, it may contain a replication origin which is a specific nucleic acid sequence at which replication is initiated. As the recombinant expression vector, various types of vectors such as plasmids, viruses, and cosmids can be used. The recombinant vector is not particularly limited as long as it expresses a desired gene in various host cells of prokaryotic and eukaryotic cells and produces a desired protein. However, it has a promoter showing strong activity and a promoter similar to a natural state A vector capable of producing a large amount of an exogenous protein in a form is preferable.

A variety of expression hosts / vectors may be used to express the antibody or fragment thereof according to the invention. Suitable expression vectors for eukaryotic hosts include, but are not limited to, SV40, cow papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus-derived expression control sequences . Expression vectors that can be used for bacterial hosts include bacterial plasmids obtained from Escherichia coli such as pET, pRSET, pBluescript, pGEX2T, pUC vector, col E1, pCR1, pBR322, pMB9 and derivatives thereof, , Phage DNA which may be exemplified by a wide variety of phage lambda derivatives such as lambda gt10 and lambda gt11, NM989, and other DNA phages such as M13 and filamentous single strand DNA phage. A useful expression vector for yeast cells is the 2 < 0 > C plasmid and its derivatives. The vector useful for insect cells is pVL941.

The recombinant vector is inserted into a host cell to form a transfection body, a suitable host cell is E. coli, Bacillus subtilis (Bacillus subtilis), genus Streptomyces (Streptomyces sp.), Pseudomonas species (Pseudomonas sp.), Proteus mirabilis , or Staphylococcus sp.). In addition, the genus Aspergillus fungi such as Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces spp. eukaryotic cells such as yeast such as Neurospora crassa , other lower eukaryotic cells, and cells of higher eukaryotes such as cells from insects.

The host cell may be derived from a plant or a mammal, preferably a monkey kidney cell (COS7), an NSO cell, a SP2 / 0, a Chinese hamster ovary (CHO) cell, a W138, a baby hamster kidney (BHK) , A myeloma cell line, a HuT 78 cell, and a HEK293 cell, but are not limited thereto. Particularly preferably, it is a CHO cell.

In the present invention, "transfection" or "transformation" into a host cell includes any method of introducing the nucleic acid into an organism, cell, tissue or organ, and selection of a suitable standard technique depending on the host cell, as is known in the art . Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, Agrobacterium mediated transfection, PEG, dextran sulfate, Pectamine and dry / inhibited mediated transfection methods, and the like.

The FcRn specific antibody according to the present invention can be produced in a large amount by culturing the transfectant expressing the recombinant vector in a nutrient medium. The medium and culturing conditions can be suitably selected depending on the host cell. The conditions such as the temperature, the pH of the medium and the incubation time can be appropriately adjusted so as to be suitable for cell growth and mass production of the protein at the time of culturing. The recombinantly produced antibody or antibody fragment can be recovered from the medium or cell lysate and can be isolated and purified by conventional biochemical separation techniques (Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd < Ed., Cold Spring Harbor Laboratory Press (1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press, Inc., San Diego, CA (1990)). Illustrative examples include, but are not limited to, electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunoadsorption chromatography, size exclusion chromatography, Method and the like can be used, but it is not limited thereto, and it is particularly preferable to separate and purify by using Protein A (Protein A).

Antigen binding ability (KD value) of the antibodies selected in the present invention was about 300 pM or less and about 2 nM or less at pH 7.4, respectively, and a KD value of 200 pM or less and 900 pM or less at pH 6.0, respectively. As described above, the antibodies selected in the present invention have a strong hFcRn binding ability of 0.01 to 2 nM at pH 6.0 and pH 7.4, so that the antibody bound outside the cell maintains binding to the endosome, thereby inhibiting the binding of the antibody to hFcRn It will be effective. These antibodies and hFcRn binding inhibitory effect were also confirmed in the inhibitory assay using FACS and a cell line expressing human FcRn.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood, however, that these embodiments are provided for purposes of illustration only and are not to be construed as limiting the scope of the present invention as defined by the appended claims. will be.

[ Example  1] Transformation Let the  Used port FcRn  Fabrication of antibody expression library

Using the transgenic Fred of the total six (OmniRat ^®, OMT, Inc.) was performed immunity (Immunization). The immunogen was human FcRn. 0.0075 mg of human FcRn was added to the soles of both sides of the rats with 8 days of immunization at 3 days intervals for 24 days with adjuvant and 28 days of immunization with 5 to 10 μg of immunogen diluted in PBS buffer. 28-day-old naled serum was taken and used for antibody titer confirmation. A 31-day old rat was euthanized and the popliteal lymph node and inguinal lymph node were recovered to fuse with P3X63 / AG8.653 myeloma cells.

ELISA analysis was performed to measure the antibody titers on the serum of the rats. First, human FcRn was diluted with PBS (pH 6.0 or pH 7.4) buffer to prepare 2 μg / mL solution, and then 100 μL of each solution was coated on a 96-well plate and left at 4 ° C. for 18 hours or more. Unbound human FcRn was removed by washing three times with 300 μL of washing buffer (0.05% Tween 20 in PBS), and 200 μL of blocking buffer was added to each well and incubated at room temperature for 2 hours. Analytical serum samples were diluted to 1/100 and the solution was diluted 2-fold in a row to prepare analytical samples with a total of 10 dilution magnifications (1/100 to 1 / 256,000). After blocking, the sample was washed three times with 300 μL of wash buffer, and the sample was allowed to stand at room temperature for 2 hours. After washing three times, the secondary antibody was diluted to 1: 50,000 in PBS buffer, and 100 μL of each antibody was added to each well. The mixture was allowed to stand at room temperature for 2 hours. After washing again 3 times, 100 μL of TMB solution was added and reacted at room temperature for 10 minutes. Then, 50 μL of 1 M sulfuric acid-containing stop solution was added to terminate the reaction, and the reaction was terminated by a microplate reader The OD value at 450 nm was measured. Immunoreactive hFcRn IgG reverse transcriptase was found to be highly immunolabile compared to the serum of untreated rats at OD 450nm of 1.0 or more at 1/100 dilution.

A total of three hybridoma libraries A, B and C were fused using polyethylene glycol. Hybridoma library A was first prepared using transgenic rats 1 and 5, hybridoma library C was used using rres 2 and 6, and hybridoma library C was used using rres 3 and 4. The fusion mixture for the preparation of each hybridoma library was cultured in a medium containing HAT for 7 days so that only cells fused by HAT were selected. Hybridoma cells surviving in HAT medium were collected and cultured for 6 days in HT medium. Then, the supernatant was taken and the amount of lead IgG was measured using a lead IgG ELISA kit (RD-biotech). First, 100 μL of each sample was diluted 1: 100, and 100 μL of each sample was added to ELISA plate well. Then, peroxidase was conjugated with labeled IgG and reacted at room temperature for 15 minutes. TMB solution was added to each well and reacted at room temperature for 10 minutes. After completion of the reaction, 50 μL of 1 M sulfuric acid-containing reaction completion solution was added, and the OD value was measured at 450 nm with a microplate reader.

[ Example  2] Hybridoma  The terms of the library hFcRn  Evaluation of Antibody Binding Ability and IgG Binding Stability of Antibodies

In order to analyze antibody binding ability to human FcRn, the same ELISA assay (pH 6.0 and pH 7.4) as the above-mentioned method was performed. The hFcRn binding capacity of the three hybridoma libraries (A, B, and C) was in the order of A> C> B library in both pH 6.0 and pH 7.4 conditions.

Evaluation of hFcRn binding using FACS at 5 ng / mL and 25 ng / mL was performed at pH 6.0 and pH 7.4 using three hybridoma library culture supernatants. Human FcRn Expression Stabilized HEK293 cells were detached from the flask and mixed in a reaction solution (0.05% BSA in PBS, pH 6.0 or pH 7.4). After diluting to a cell number of 2 x 10 ⁶ cells / mL, 50 μL of the supernatant was added to 96 wells, and 50 μL of the hybridoma library culture supernatant diluted to 10 ng / mL and 50 ng / mL was added to the wells. Lt; / RTI > A488 rabbit anti-rIgG goat Ab was diluted 1: 200 in the reaction solution, and 100 μL of the solution was added to the cell pellet. The reaction mixture was mixed with 150 μL of the reaction solution, and the result was read by FACS (BD). As with the ELISA results, it was confirmed that the hybridoma library A had the highest binding affinity.

Evaluation of human FcRn inhibition of hybridoma library using FACS was carried out at pH 6.0. First, naive HEK293 cells and human FcRn-overexpressing HEK293 cells were mixed in a reaction solution (0.05% BSA in PBS, pH 6.0). 1 × 10 ⁵ cells were placed in 96 wells and each hybridoma library culture supernatant was treated with 4 nM and a 0.4 nM concentration diluted 10-fold. To confirm the inhibition of hIgG binding, 100 nM A488-hIgG1 was added to each well and reacted on ice for 90 minutes. At the end of the reaction, the cell pellet was washed with 100 μL of the reaction solution, transferred to a U-shaped bottom test tube, and measured by FACS. The amount of 100 nM A488-hIgG1 remaining in human FcRn overexpressing stabilized cells was measured, and the binding inhibition (%) was determined. HIgG1 was used as a reference control (Isotype control), and the antibody binding inhibition ability was compared using a previously developed HL161-1Ag antibody as a positive control. Each control was analyzed at concentrations of 1 μM and 2 μM, and hybrid library samples were measured at both concentrations of 0.4 nM and 4 nM. As a result, it was confirmed that the hybridoma library A showed the highest binding inhibitory effect.

[ Example  3] FACS  Used Hybridoma  Isolate cell clones and secure human antibodies

Clones were isolated by FACS using hybridoma library A, which exhibited the highest human FcRn binding capacity and inhibitory activity, resulting in a total of 442 single clones. The separated clones were incubated with HT medium, and the supernatant was collected to select antibody-expressing hybridomas binding to hFcRn with FACS. As a result, 100 clones (M1-M100) were strongly bound to hFcRn expressing HEK293 cells .

Sequence analysis was performed by separating RNAs of 100 single clones selected by FACS analysis. From the first sequence analysis, 88 out of 100 sequences were analyzed and divided into 35 groups (G1 to G38) based on the amino acid sequence. The culture supernatants of 33 representative clones were diluted to a concentration of 100 ng / mL and the binding force to hFcRn was evaluated by ELISA, except for two clones (G33, G35) in which the culture was not secured.

Evaluation of hFcRn binding capacity using FACS was performed at pH 6.0 and 7.4 as in the previous experiment. The ranking according to the degree of binding between clones was similarly analyzed by pH, and the intensity of binding was varied.

In addition, the inhibition of hFcRn was analyzed using FACS at pH 6.0 for the above 33 clones. The inhibitory activity (%) was calculated based on the measured MFI value. A group of 70 ~ 100% was classified as B, a group of 30 ~ 70% was classified as B, a group of 10 ~ 30% was classified as C, a group as 10% or less D, respectively.

Kinetic analysis of the hybridoma clone using SPR was carried out by immobilizing human FcRn and then using hybridoma medium as an assay sample. Most, except for some clones clones showed more than 10 ⁶ M value kon, koff values are 10 ^- was determined to be less than ³ M. In conclusion, all clones were found to have a KD value of 10 ^-9 to 10 ^-11 M.

The genes of 18 clones that do not contain N-glycosylation or free cysteine in the CDR sequence region of antibody sequences of A and B groups divided by the result of hFcRn inhibition among 35 kinds of hybridomas clones are converted into complete human IgG sequences Respectively.

First, the amino acid sequence homology of the 18 selected antibodies VH and VL with the human antibody gullet line antibody group can be found on the NCBI web page (http://www.ncbi.nlm.nih.gov/igblast/) Ig BLAST program.

To clone 18 human antibody genes, restriction enzyme recognition sites were inserted at both ends of the gene as follows. The EcoRI / NheI restriction enzyme recognition site was designed for the heavy chain variable region (VH) and EcoRI / Xhol for the light chain variable region (VL (λ)) and the EcoRI / NheI restriction enzyme recognition region for the light chain variable region (VL In the case of the variable region (VL), the light chain lambda variable (VL (lambda)) gene sequence in gene cloning is a human light chain constant (LC) constant region gene and the light chain kappa variable (VL And linked to the light chain constant (LC (kappa) constant region) gene.

Cloning of pCHO1.0 expression vector for antibody expression in animal cells was performed by digesting light and heavy chain genes with restriction enzymes EcoRV, PacI, AvrII and BstZ17I, respectively. A total of 18 confirmed human antibody gene-containing pCHO1.0 expression vectors were subjected to DNA sequencing to confirm that they were identical to the synthetic gene sequence.

The expression vector pCHO1.0 expressed a complete human IgG form as an animal cell expression system containing both an antibody light chain and a heavy chain gene. Human antibodies were obtained by transfecting plasmid DNA of each of the above antibodies into CHO-S cells and purifying the antibody secreted in the medium using Protein A column.

 human IgG was injected into Tg32 (hFcRn + / +, h? 2m + / +, mFcRn- / -, m? 2m - / -) mice (Jackson Laboratory) in which hFcRn was expressed and then 18 human antibodies converted into human IgG sequences were administered Human IgG.

Affinity (KD) and human using a FACS FcRn binding affinity and inhibitory ability analysis of in vitro human 4 the most effective action in the catabolism of in vivo human IgG species using the analysis result and the Tg32 mouse anti for the foregoing antigen FcRn antibody, HL161A , HL161B, HL161C and HL161D antibody proteins (Fig. 1). In addition, the HL161BK antibody was obtained by removing the N-glycosylation site by substituting Lysine (K) for the 83rd Asparagine (N) of the heavy chain variable framework framework of the HL161B antibody. The gene sequences, amino acid sequences and CDR sequences of each of these light and heavy chain variable regions are shown in Tables 1, 2 and 3 below.

Table 1. Selected human FcRn  Antibody Heavy chain  And Light chain  The polynucleotide sequence of the variable region

Table 2. Selected human FcRn  Antibody Heavy chain  And Light chain  The amino acid sequence of the variable region

Table 3. Selected human FcRn  Antibody Heavy chain  And Light chain  Variable area CDR  order

[ Example  4] HL161A / HL161B / HL161C / HL161D SPR  Antibody binding assay

The binding affinity of HL161A, HL161B, HL161C, and HL161D antibodies via SPR was measured by immobilizing soluble hFcRn as a ligand on a Proteon GLC chip (Bio-Rad). Kinetic analysis was performed using a Proteon XPR36 instrument, and shFcRn was immobilized on a GLC chip, and antibody samples were reacted at 5 concentrations to obtain a sensogram result. Kinetic analysis was performed using a 1: 1 Langmuir binding model, and the mean KD value was determined by repeating six times at pH 6.0 and pH 7.4. Activation was performed by EDAC / NHS 0.5X, 30 μL / min, 300 sec, and the immobilization was performed using 2 μg / mL and 250 μL of shFcRn using acetate buffer (pH 5.5) And the reaction was stopped when the immobilization level was 200 to 300 RU while flowing at 30 μL / min. The inactivation was then carried out at 30 μL / min for 300 seconds using ethanolamine. Each HL161 antibody was diluted stepwise by 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, and 0.312 nM based on 10 nM concentration. Sample dilution was performed using 1X PBST (pH 7.4) or 1X PBST (pH 6.0) buffer depending on the assay pH. The sample was allowed to react at 50 μL / min for 200 sec, and the dissociation step was performed at 50 μL / min for 600 sec and then regeneration was performed at 100 μL / min for 18 sec using glycine buffer (pH 2.5) Respectively. After six kinetic analyzes per sample, the average antigen binding (KD) was determined. The kinetic parameters of the antibodies resulting from the SPR analysis are shown in Table 4 (Figures 2a to 2h).

Table 4. Human FcRn  Immobilization SPR  Antibodies used Kinetic  Analysis

[ Example  5] HL161A / HL161B FACS  Human antibodies used FcRn  Bond analysis

HEK293 - stabilized cells expressing human FcRn were analyzed for FcRn binding by pH using FACS system. The FcRn binding assay using FACS was carried out at pH 6.0 and pH 7.4 according to the pH to be evaluated. First, 100,000 human FcRn stabilized HEK293 cells were washed with PBS buffer and centrifuged at 4500 rpm for 5 minutes in a tabletop microcentrifuge to make cell pellets. Antibodies were added to 100 μL of pH 6.0 or pH 7.4 PBS / 10 mM EDTA. The reaction solution was added to the remaining cell pellets, and cell counting was performed. 10 μL of the cell suspension was put on a slide, and the number of cells was counted in a TC10 instrument and diluted with the reaction solution to 2 × 10 ⁶ cells / mL. Each antibody sample was diluted to 500 nM, diluted to 20 nM on a 96-well v-bottom plate for pH 6.0 analysis, and 50 μl aliquots were added to each well. For pH 7.4 analysis, 500 nM antibody samples were diluted by 3 fold serial dilution and analyzed at final concentrations ranging from 250 nM to 0.11 nM. Cells diluted to 2 x 10 ⁶ cells / mL were added in a volume of 50 μL. The plate was placed on a rotator installed at 4 ° C and rotated at 15 ° angle for 10 minutes at 10 rpm. At the end of the reaction, the plate was taken out, centrifuged at 2000 rpm for 10 minutes, and the supernatant was removed. A488 anti-hIgG Goat Ab was diluted 1: 200 in the reaction solution, and 100 μL of each was added to each well. Again, the plate was placed on a rotator installed at 4 ° C and rotated for 90 minutes at 15 degrees and 10 rpm. At the end of the reaction, the plate was taken out, centrifuged at 2000 rpm for 10 minutes, and the supernatant was removed. After this washing process, 100 μL of the reaction solution was added to the cell pellet, and the cell pellet was transferred to a blue test tube. 200 占 퐇 of the reaction solution was added thereto, and the measurement was read in FACS. The FACS measurement conditions are FS 108 volts, SS 426 volts, FL1 324 volts, and FL2 300 volts. These cells were analyzed by FACS using BD FACSDiva ^™ v6.1.3 software (BD Bioscience). The results were expressed as Mean Fluorescence Intensity (MFI) (Figure 3). The HL161A and HL161B antibodies exhibited MFI values of 10.59 and 8.34 at 10 nM and pH 6.0, respectively, and 4 parameter logistic regression using MFI values at pH 7.4 at 0.11 to 250 nM concentration. The results of EC50 (Effective Concentration 50%) were 2.46nM and 1.20nM, respectively.

[ Example  6] HL161A / HL161B FACS  Of antibody used Inhibition  Function analysis

Two kinds of antibodies that had been analyzed for binding ability to human FcRn on the cell surface were treated with HEK293 cells expressing hFcRn on the cell surface, and the binding of Alexa-Fluo-488 fluorescently labeled hIgG1 was decreased to confirm the inhibitory effect of the antibody Respectively. The analysis process is described in detail as follows.

Na 2 HEK 293 cells and human FcRn overexpression-stabilized HEK 293 cells were each put in 2 ml of 1 x TE and incubated for 1 minute at 37 ° C in a 5% CO2 incubator. After recovering the cells from the flask, 8 mL of pH 6.0 reaction solution was added and mixed and transferred to a 50 mL cornical tube. After centrifugation at 2000 rpm for 5 minutes, the supernatant was removed, and 1 mL of pH 6.0 reaction solution was added to each cell pellet, and the mixture was transferred to a new 1.5 mL Eppendorf tube. After centrifugation at 4000 rpm for 5 minutes, the supernatant was removed, and the remaining cell pellet was added with pH 6.0 reaction solution, and the cell number of the cell suspension was measured. Finally, the cell suspension was diluted with the reaction solution to 2.5 x 10 ⁶ cells / mL.

Each antibody sample was diluted to 400 nM and then diluted 4-fold in 96-well v-bottom plates. Samples diluted to a final concentration of 200 nM to 0.01 nM were dispensed into each well in an amount of 50 μL. 10 μL of Alex488-hIgG1 diluted in the pH 6.0 reaction solution was dispensed at 1 μM. Finally, 40 μL of cells diluted to 2.5 × 10 ⁶ cells / mL were added and mixed. The plate was placed on a rotator installed at 4 ° C and rotated for 90 minutes at 15 rpm and 10 rpm. When the reaction was completed, the plate was taken out, centrifuged at 2000 rpm for 10 minutes, and the supernatant was removed. The cell pellet was resuspended in 100 μL of the reaction solution, transferred to a blue test tube, 200 μL of the reaction solution was added, and the result was read by FACS. The FACS measurement conditions are FS 108 volts, SS 426 volts, FL1 324 volts, and FL2 300 volts. These cells were analyzed by FACS using BD FACSDiva ^™ v6.1.3 software (BD Bioscience). The results were expressed as mean fluorescence intensity (MFI). The MFI of the experimental group was subtracted from the MFI value (background signal) measured only through the cells and then treated. The MFI of the control tube (Alexa Fluor 488 alone and no competition factors) was converted to 100% and the percentage of the MFI of the competitor-containing tube was calculated.

The competition ratio of competition antibody was higher when the concentration of the competition antibody was lower than the MFI of the human IgG1 competent factor-containing tube. The inhibition rate of HL161A and HL161B antibody was measured in the range of 0.01 ~ 200nM at pH 6.0, Logistic regression analysis was performed.

As a result, IC50 (inhibitory concentration 50%) values of HL161A and HL161B antibodies were 0.92 nM and 2.24 nM, respectively (FIG. 4).

[ Example 7 ] mFcRn - / - hFCRN  Transformation 32 ( Tg32 ) From the mouse HL161A / HL161B  Effectiveness test

Human IgG was injected into Tg32 (hFcRn + / +, h? 2m + / +, mFcRn- / -, m? 2m - / -) mice (Jackson Laboratory) in which human FcRn was expressed and then HL161A and HL161B and human IgG were administered, And whether it affects the action.

HL161A and HL161B antibodies and human IgG (Greencross, IVglobulinS) were kept at 4, 5, 10, and 20 mg / kg doses for 4 days and PBS buffer at pH 7.4 was used as the vehicle and 20 mg / kg IgG1 control . Human FcRn Tg32 mice were adapted for about 7 days and were allowed free access to water and feed and were automatically controlled for temperature (23 ± 2 ° C), humidity (55 ± 5%) and darkness (12 hours) . Experiments were carried out on 4 mice per group. Biotin conjugated hIgG was prepared using a kit (Pierce, Cat #. 21327) to use human IgG as a tracer. At 0 hour, 5 mg / kg of biotin-hIgG and 495 mg / kg of human IgG were intraperitoneally administered to saturate IgG in the body. The drug was administered intraperitoneally once daily at a dose of 5, 10 and 20 mg / kg at 24, 48, 72 and 96 hours after the administration of biotin-IgG. Blood sampling was performed by light anesthesia using Isoflurane (JW Pharmaceutical), and then blood was collected from the orbital vein using a heparinized micro-hematocrit capillary tube (Fisher). 24, 48, 72, 96, 120, and 168 hours after the administration of biotin-IgG and 24, 48, 72, and 96 hours after the administration of the biotin-IgG. After receiving 0.1 mL of whole blood in an Eppendorf tube, the plasma was separated by a centrifuge immediately and stored at -70 ° C in a deep freezer (Thermo) until analysis.

The amount of biotin-hIgG1 in the collected blood was analyzed by ELISA as follows. 100 μL of neutravidin (Neutravidin, Pierce, 31000) was added to a 96-well plate (Costar, Cat. No. 2592) at a concentration of 1.0 μg / ml and fixed at 4 ° C. for 16 hours.

The plates were washed three times with buffer A (0.05% Tween-20, 10 mM PBS, pH 7.4), and then reacted in PBS (pH 7.4) containing 1% BSA at room temperature for 2 hours. After washing the plate three times with buffer A, a neutravidin plate was prepared with PBS (pH 7.4) solution containing 0.5% BSA so as to correspond to 1 μg / ml. Blood samples were sequentially diluted in the range of 500 to 1000 times by using buffer B (100 mM MES, 150 mM NaCl, 0.5% BSA IgG-free, 0.05% Tween-20, pH 6.0) Lt; / RTI > The injected samples were reacted at room temperature for 1 hour. After washing the plate three times with buffer A, HRP-conjugated anti-human IgG goat antibody was prepared at 1 nM, 200 μL of each antibody was added, and reacted at 37 ° C. for 2 hours. After washing the plate three times with ice buffered buffer B, 100 μL of 3,3 ', 5,5'-tetramethylbenzidine (RnD, Cat. No: DY999) as a substrate solution was injected The reaction was carried out at room temperature for 15 minutes. The reaction was stopped by injecting 50 μL of a 1.0 M sulfuric acid solution (Seonjeon, Cat. No: S2129), and the absorbance at 450 nm was measured.

The concentration of biotin-IgG was fixed at 100% after 24 hours (the approximate Tmax of biotin-IgG in mouse and the time before biotin-IgG catabolism occurred), and the concentration of the remaining time was expressed in% Is shown in FIG. The half-lives of the vehicle and the 20 mg / kg IgG1 control were 103 and 118 hours, respectively. On the other hand, the in vivo half-life of IgG of human HL161A antibody, which showed the fastest IgG catabolism in human FcRn transgenic Tg32 mouse, was superior to human FcRn binding ability and inhibitory ability in the in vitro analysis, and was 30, 23 and 18 hours respectively. In addition, the HL161B antibody showed IgG half-life of 41, 22, and 21 hours, respectively, by dose. This confirmed that pH-independent, Fc non-competitive antibody to hFcRn was effective in increasing the catabolism of endogenous antibodies (FIGS. 5A and 5B).

[ Example  8] from a monkey HL161A / HL161B  Effectiveness test

Monkey IgG, IgA, IgM, and albumin levels by administration of HL161A and HL161B antibody were confirmed and the pharmacokinetic (PK) profile was confirmed using a Cynomolgus monkey having 96% homology with human FcRn.

1) Monkeys In the blood IgG  Analysis of antibody changes

First, monkey IgG was assayed for changes through ELISA analysis. 100 μL of anti-human IgG Fc antibody (BethylLab, A80-104A) was loaded onto a 96 well plate (Costar, Cat. No. 2592) at a concentration of 4.0 μg / mL and fixed at 4 ° C. for 16 hours. The plates were washed three times with washing solution (0.05% Tween-20, 10 mM PBS, pH 7.4) and then reacted in PBS (pH 7.4) containing 1% BSA for 2 hours at room temperature. Blood samples were diluted 80,000 times with PBS (pH 7.4) containing 1% BSA, loaded on a plate, and reacted at room temperature for 2 hours. After washing the plate three times with washing solution, 100 μL of the antibody against hIgG (Biorad, 201005) was diluted 20,000 times, and reacted at room temperature for 1 hour. After washing each plate, 100 μL of 3,3 ', 5,5'-tetramethylbenzidine (RnD, Cat. No: DY999) as a substrate solution was injected, reacted at room temperature for 7 minutes, and 1.0 M sulfuric acid solution , Cat. No: S2129) was injected to stop the reaction. The absorbance (OD) was measured using a 450 and 540 nm absorbance reader (MD, Model: VersaMax). As a result, intravenous administration of HL161A and HL161B antibody to Cynomolgus monkeys at a dose of 5, 20 mg / kg once a week resulted in a dose-dependent decrease in monkey IgG dose-dependently and the IgG-FcRn interaction was effectively inhibited by the HL161 antibody . In the case of 5 mg / kg of HL161A, it was reduced to 47.1% on the 9th day and decreased to 29.6% on the 10th day by 20mg / kg. 5 mg / kg of HL161B was reduced to 53.6% at 10 days, and 20 mg / kg was reduced to 31% at 9 days, with similar results in both antibodies (Table 5 and Figures 6a-6c) . In addition, the changes of monkey IgG by intravenous administration of HL161A and HL161B were compared with each other, and the monkey IgG tended to decrease very much in each individual.

Table 5. HL161A and HL161B  Monkeys by administration Of IgG  change( % )

2) Monkeys In the blood HL161A / HL161B  Pharmacokinetic profile ( Pharmacokinetic  profile analysis results

Serum pharmacokinetic profiles over time following intravenous administration of HL161A and HL161B were analyzed using competitive ELISA methods. First, a 2 μg / mL solution of Neutravidin was prepared, and 100 μL of each solution was coated on a 96-well plate, followed by standing at 4 ° C. for 18 hours. After washing three times with 300 μL of wash buffer (10 mM PBS, pH 7.4 with 0.05% Tween 20), each well was reacted with PBS (pH 7.4) solution containing 1% BSA at 25 ° C for 2 hours at room temperature. Biotin-conjugated hFcRn was diluted with PBS to make 1 μg / mL solution. 100 μL of each solution was dispensed into a 96-well plate and reacted at 25 ° C for 1 hour. The unbound hFcRn was removed by washing with 300 μL of wash buffer three times, and then the reference sample (0.156 to 20 ng / mL) was added and reacted at 25 ° C. for 2 hours. After washing three times with washing buffer, the detection antibody was diluted to 1: 10,000 in PBS, and 100 μL of the diluted antibody was added to each well. The reaction was carried out at 25 ° C. for 1.5 hours. After washing three times, 100 μL of TMB solution was added. After reacting at room temperature for 5 minutes, 50 μL of 1 M sulfuric acid-terminated solution was added and the reaction was terminated. The absorbance at 450 nm was measured with a absorbance reader. The results of analysis of HL161A and HL161B are shown in Table 6, and it was confirmed that the dose-dependent increase was observed. The half-life (T1 / 2) was about 6 days to 12 days, which was shorter than the half-life of antibodies generally known. Overall, HL161B had a half-life, AUC and Cmax higher than HL161A (Figs. 7A and 7B).

Table 6. HL161A and HL161B  Results of pharmacokinetic profile analysis by dose

3) Monkeys In the blood IgM  And IgA  Analysis of antibody changes

ELISA analysis for measuring IgM and IgA in monkey blood proceeded similar to ELISA for IgG measurement. First, 100 μL anti-monkey IgM antibody (Alpha Diagnostic, 70033) or IgA antibody (Alpha Diagnostic, 70043) was dispensed in a 96-well plate at a concentration of 2.0 μg / mL and immobilized at 4 ° C. for 16 hours. The plate was washed three times with washing solution (10 mM PBS containing 0.05% Tween-20, pH 7.4), and then reacted in PBS (pH 7.4) containing 1% BSA for 2 hours at room temperature. The blood samples were analyzed by using PBS (pH 7.4) solution containing 1% BSA in a concentration range of 7.8 to 1,000 ng / mL and monoclonal IgM in the range of 15.6 to 2,000 ng / mL. After dilution, the cells were added to each well and reacted at room temperature for 2 hours. The plate was washed three times with the washing solution, and then the secondary antibody (Alpha Diagnostic, 70031) against monkey IgM and the secondary antibody (KPL, 074-11-011) against monkey IgA were diluted 5,000 times, The reaction was allowed to proceed at room temperature for 1 hour. After washing three times, 100 μL of 3,3 ', 5,5'-tetramethylbenzidine (RnD, Cat. No: DY999) was injected and reacted at room temperature for 7 minutes. A 1.0 M sulfuric acid solution (Cat. No. S2129), and the absorbance was measured using a 450, 540 nm absorbance reader (Model: VersaMax).

4) Monkeys In the blood  Analysis results of albumin change

Analysis of the amount of monkey albumin change in blood was analyzed using a commercially available ELISA kit (Assaypro, Cat. No: EKA2201-1). To summarize the method, monkey sera, which is an analytical sample, was diluted 4,000 times, and 25 μL aliquots were added to a 96-well plate immobilized with monkey albumin. 25 μL of biotin-conjugated monkey albumin solution was added thereto, followed by reaction at 25 ° C. for 2 hours. After washing three times with 200 μL of wash buffer, 50 μL of a 1: 100 dilution of a secondary antibody conjugated with streptavidin-peroxidase was added to each well and reacted at 25 ° C. for 30 minutes. After washing three times, 50 μL of the substrate was added and reacted at room temperature for 10 minutes. Then, 50 μL of the reaction-terminated solution was added and the absorbance at 450 nm was measured. In conclusion, monkey IgM, IgA, and albumin by HL161A and HL161B antibody administration did not show any significant change during the entire test period (FIGS. 8A to 8C). Therefore, it was concluded that HL161 antibody is only involved in IgG, and it does not affect the IgM and IgA concentrations, and it is considered that the immunoglobulin concentration is less affected by the decrease of immunoglobulin concentration. In addition, monkey albumin concentration did not change significantly during the entire study period, indicating that HL161A and HL161B antibodies specifically blocked IgG-FcRn interactions.

5) Blood biochemical and urine composition analysis

Finally, blood biochemical test and urine test by antibody administration were carried out using a sample on the 14th day of the test. Blood biochemical markers are aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine phosphokinase (CPK), total bilirubin (TBIL), glucose (GLU), total cholesterol (TCHO), triglyceride ), Total protein (TP), Alumine (Alb), Albumine / globulin (A / G), Blood urea nitrogen (BUN), Creatinine (CRE), Inorganic phosphorus (IP), Calcium (Ca), Natrium (LEU), Nitrate (NIT), Urobilinogen (URO), Protein (PRO), pH, and Occult blood (URO) were analyzed using a Hitachi 7180 instrument. (GLO), ascorbic acid (ASC) were measured using a Mission U120 instrument. The results were as follows: 1. Overall, there were some numerical changes, but they were measured to be within the normal range of the Cynomolgus monkey.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

&Lt; 110 > Hanall Biopharma Co., Ltd. <120> FcRn Specific Human Antibody and Composition for Treatment of          Autoimmune Diseases <130> 250 &Lt; 150 > US 61 / 986,742 <151> 2014-04-30 <160> 44 <170> Kopatentin 2.0 <210> 1 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Hv of HL161A <400> 1 gaagtgcagc tgctggaatc cggcggaggc ctggtgcagc ctggcggctc tctgagactg 60 tcctgcgccg cctccgagtt caccttcggc agctgcgtga tgacctgggt ccgacaggct 120 cccggcaagg gcctggaatg ggtgtccgtg atctccggct ccggcggctc cacctactac 180 gccgactctg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240 ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc caagaccccc 300 tggtggctgc ggtccccctt cttcgattac tggggccagg gcaccctggt gacagtgtcc 360 tcc 363 <210> 2 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Hv of HL161A <400> 2 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Gly Ser Cys              20 25 30 Val Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Val Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Lys Thr Pro Trp Trp Leu Arg Ser Pro Phe Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 3 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Hv of HL161B <400> 3 caactgttgc tccaggaatc cggtcctggt cttgtaaagc catctgagac tctctccctt 60 acctgtaccg ttagcggagg aagtctttcc tcaagcttct cctactgggt gtggatcaga 120 cagcctcccg gaaaagggtt ggagtggatt ggcacaatat actactccgg caacacttac 180 tataacccca gcctgaagag caggctgact atctctgtcg acaccagtaa aaatcacttt 240 tctctgaatc tgtcttcagt gaccgcagcc gacaccgccg tgtattattg cgctcggcgc 300 gccgggattc tgacaggcta tctggattca tggggccagg ggacattggt tacagtgtct 360 agt 363 <210> 4 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Hv of HL161B <400> 4 Gln Leu Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu   1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Ser Ser Ser              20 25 30 Phe Ser Tyr Trp Val Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu          35 40 45 Trp Ile Gly Thr Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser      50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn His Phe  65 70 75 80 Ser Leu Asn Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr                  85 90 95 Cys Ala Arg Arg Ala Gly Ile Leu Thr Gly Tyr Leu Asp Ser Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 5 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Hv of HL161BK <400> 5 cgctgctgc tgcaagaatc cggccctggc ctggtgaaac cctccgagac actgtccctg 60 acctgcaccg tgtccggcgg ctccctgtcc tccagcttct cctactgggt ctggatccgg 120 cagccccctg gcaagggcct ggaatggatc ggcaccatct actactccgg caacacctac 180 tacaacccca gcctgaagtc ccggctgacc atctccgtgg acacctccaa gaaccacttc 240 agcctgaagc tgtcctccgt gaccgccgct gacaccgccg tgtactactg tgccagaagg 300 gccggcatcc tgaccggcta cctggactct tggggccagg gcaccctggt gacagtgtcc 360 tcc 363 <210> 6 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Hv of HL161BK <400> 6 Gln Leu Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu   1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Ser Ser Ser              20 25 30 Phe Ser Tyr Trp Val Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu          35 40 45 Trp Ile Gly Thr Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser      50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn His Phe  65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr                  85 90 95 Cys Ala Arg Arg Ala Gly Ile Leu Thr Gly Tyr Leu Asp Ser Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 7 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> Hv of HL161C <400> 7 caggtgcagc tcgtgcagtc cggcgcagag gtcaaaaagc ctggtgcatc tgtgaaagtg 60 agttgcaagg ctagcggcta cacctttacc ggatgttata tgcattgggt acgccaagcc 120 cccggacaag gcttggaatg gatggggcgt atcaacccaa actctggcgg gactaattac 180 gcccagaagt ttcagggaag ggtgactatg acaagggaca catccatatc caccgcttat 240 atggacctgt ctcgactgcg gtctgatgat acagccgttt attactgcgc cagagactac 300 agcggatgga gcttcgatta ttgggggcag ggtactttgg tcacagtttc aagt 354 <210> 8 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Hv of HL161C <400> 8 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Cys              20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr  65 70 75 80 Met Asp Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Asp Tyr Ser Gly Trp Ser Phe Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 9 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Hv of HL161D <400> 9 cagctgcagt tgcaggagtc aggccccggt ttggttaagc cttctgaaac cctttctctc 60 acatgcacag tatccggtgg ctccatctcc agttcaagtt actactgggg atggatccgg 120 caacccccag gaaaagggct ggagtggatt ggcaatatat attactctgg gtccacctat 180 tacaaccctt ccctgatgag tagagtgacc atcagcgtgg acacaagcaa aaaccaattc 240 agcctgaagc tttctagcgt gaccgctgcc gacacagctg tctattactg tgcccgccag 300 cttagttata actggaatga taggctgttt gattactggg gccaggggac tctcgttaca 360 gtcagcagc 369 <210> 10 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Hv of HL161D <400> 10 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu   1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ser Ser Ser Ser              20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu          35 40 45 Trp Ile Gly Asn Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser      50 55 60 Leu Met Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe  65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr                  85 90 95 Cys Ala Arg Gln Leu Ser Tyr Asn Trp Asn Asp Arg Leu Phe Asp Tyr             100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 11 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Lv of HL161A <400> 11 tcttacgtgc tgacccagcc cccctccgtg tctgtggctc ctggccagac cgccagaatc 60 acctgtggcg gcaacaacat cggctccacc tccgtgcact ggtatcagca gaagcccggc 120 caggcccccg tgctggtggt gcacgacgac tccgaccggc cttctggcat ccctgagcgg 180 ttctccggct ccaactccgg caacaccgcc accctgacca tctccagagt ggaagccggc 240 gacgaggccg actactactg ccaagtgcga gactcctcct ccgaccacgt gatcttcggc 300 ggaggcacca agctgaccgt gctgggccag cctaaggccg ctccctccgt gaccctg 357 <210> 12 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Lv of HL161A <400> 12 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln   1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Thr Ser Val              20 25 30 His Trp Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val His          35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser      50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly  65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Arg Asp Ser Ser Ser Asp His                  85 90 95 Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys             100 105 110 Ala Ala Pro Ser Val Thr Leu         115 <210> 13 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Lv of HL161B <400> 13 tcttacgtgc tgacccagtc cccctccgtg tccgtggctc ctggccagac cgccagaatc 60 acctgtggcg gcaacaacat cggctccaag tccgtgcact ggtatcagca gaagcccggc 120 caggcccccg tgctggtggt gtacgacgac tccgaccggc cctctggcat ccctgagcgg 180 ttctccgcct ccaactccgg caacaccgcc accctgacca tctccagagt ggaagccggc 240 gacgaggccg actactactg ccaagtgtgg gactcctcct ccgaccacgt ggtgttcggc 300 ggaggcacca agctgaccgt gctgggccag cctaaggccg ctccctccgt gaccctg 357 <210> 14 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Lv of HL161B <400> 14 Ser Tyr Val Leu Thr Gln Ser Pro Ser Val Ser Val Ala Pro Gly Gln   1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val              20 25 30 His Trp Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr          35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Ala Ser      50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly  65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His                  85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys             100 105 110 Ala Ala Pro Ser Val Thr Leu         115 <210> 15 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Lv of HL161BK <400> 15 tcttacgtgc tgacccagtc cccctccgtg tccgtggctc ctggccagac cgccagaatc 60 acctgtggcg gcaacaacat cggctccaag tccgtgcact ggtatcagca gaagcccggc 120 caggcccccg tgctggtggt gtacgacgac tccgaccggc cctctggcat ccctgagcgg 180 ttctccgcct ccaactccgg caacaccgcc accctgacca tctccagagt ggaagccggc 240 gacgaggccg actactactg ccaagtgtgg gactcctcct ccgaccacgt ggtgttcggc 300 ggaggcacca agctgaccgt gctgggccag cctaaggccg ctccctccgt gaccctg 357 <210> 16 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Lv of HL161BK <400> 16 Ser Tyr Val Leu Thr Gln Ser Pro Ser Val Ser Val Ala Pro Gly Gln   1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val              20 25 30 His Trp Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr          35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Ala Ser      50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly  65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His                  85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys             100 105 110 Ala Ala Pro Ser Val Thr Leu         115 <210> 17 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> Lv of HL161C <400> 17 gacatccaga tgacccagtc accatcatcc ctttccgcat ctgtcggaga tagagtgact 60 atcacctgca gggcttctca aggtatttcc aactacctcg cctggttcca gcaaaagcca 120 ggtaaagccc caaagagctt gatctacgcc gcttctagtc tgcagagtgg agttcctagt 180 aagttctccg gctctggcag tggcacagat tttaccttga ccatttccag cctgcagtct 240 gaggatttcg ctacctacta ttgtcagcag tatgacagct atccccccac atttgggggg 300 ggcactaagg tggagataaa acggacagtg gctgcccctt ctgtctttat t 351 <210> 18 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Lv of HL161C <400> 18 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr              20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile          35 40 45 Tyr Ala Ser Ser Leu Gln Ser Ser Val Ser Ser Ser Val Ser Ser      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser  65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Tyr Pro Pro                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile         115 <210> 19 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> Lv of HL161D <400> 19 agctatgagc tgacccagcc tctgagcgta tctgtcgctc tcggccagac agccagaatt 60 acctgtggcg gcaataacat aggatccaaa aatgttcact ggtatcagca aaaacctggc 120 caagctcccg tgctcgtgat ctaccgggac tctaaccgac ccagtggaat ccccgaacgc 180 tttagcggtt ccaactctgg aaatacagct actctgacta tctccagggc tcaggccggg 240 gatgaggccg attactactg ccaggtgtgg gactcaagca cagtggtctt cggcggaggt 300 accaagttga ctgttcttgg gcagccaaag gccgcacctt cagtgaccct g 351 <210> 20 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Lv of HL161D <400> 20 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln   1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val              20 25 30 His Trp Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr          35 40 45 Arg Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser      50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly  65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Thr Val Val                  85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala             100 105 110 Pro Ser Val Thr Leu         115 <210> 21 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Hv CDR1 of HL161A <400> 21 Ser Cys Val Met Thr   1 5 <210> 22 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Hv CDR2 of HL161A <400> 22 Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 23 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Hv CDR3 of HL161A <400> 23 Thr Pro Trp Trp Leu Arg Ser Pro Phe Phe Asp Tyr   1 5 10 <210> 24 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Lv CDR1 of HL161A <400> 24 Gly Asn Asn Ile Gly Ser Thr Ser Val His   1 5 10 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Lv CDR2 of HL161A <400> 25 Asp Asp Ser Asp Arg Pro Ser   1 5 <210> 26 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Lv CDR3 of HL161A <400> 26 Val Arg Asp Ser Ser Ser Asp His Val Ile   1 5 10 <210> 27 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Hv CDR1 of HL161B or HL161BK <400> 27 Phe Ser Tyr Trp Val   1 5 <210> 28 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Hv CDR2 of HL161B or HL161BK <400> 28 Thr Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser Leu Lys Ser   1 5 10 15 <210> 29 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Hv CDR3 of HL161B or HL161BK <400> 29 Arg Ala Gly Ile Leu Thr Gly Tyr Leu Asp Ser   1 5 10 <210> 30 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Lv CDR1 of HL161B or HL161BK <400> 30 Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His   1 5 10 <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Lv CDR2 of HL161B or HL161BK <400> 31 Asp Asp Ser Asp Arg Pro Ser   1 5 <210> 32 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Lv CDR3 of HL161B or HL161BK <400> 32 Gln Val Trp Asp Ser Ser Ser Asp His Val   1 5 10 <210> 33 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Hv CDR1 of HL161C <400> 33 Gly Cys Tyr Met His   1 5 <210> 34 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Hv CDR2 of HL161C <400> 34 Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln   1 5 10 15 Gly     <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Hv CDR3 of HL161C <400> 35 Asp Tyr Ser Gly Trp Ser Phe Asp Tyr   1 5 <210> 36 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Lv CDR1 of HL161C <400> 36 Arg Ala Ser Gln Gly Ile Ser Asn Tyr Leu Ala   1 5 10 <210> 37 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Lv CDR2 of HL161C <400> 37 Ala Ala Ser Ser Leu Gln Ser   1 5 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Lv CDR3 of HL161C <400> 38 Gln Gln Tyr Asp Ser Tyr Pro Pro Thr Phe   1 5 10 <210> 39 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Hv CDR1 of HL161D <400> 39 Ser Tyr Tyr Trp Gly   1 5 <210> 40 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Hv CDR2 of HL161D <400> 40 Asn Ile Tyr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Sel   1 5 10 15 <210> 41 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Hv CDR3 of HL161D <400> 41 Gln Leu Ser Tyr Asn Trp Asn Asp Arg Leu Phe Asp Tyr   1 5 10 <210> 42 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Lv CDR1 of HL161D <400> 42 Gly Gly Asn Asn Ile Gly Ser Lys Asn Val His   1 5 10 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Lv CDR2 of HL161D <400> 43 Arg Asp Ser Asn Arg Pro Ser   1 5 <210> 44 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Lv CDR3 of HL161D <400> 44 Gln Val Trp Asp Ser Ser Thr Val Val   1 5

Claims (25)

CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 42;
CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40 and SEQ ID NO: And
A CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: -FcRn antibody or fragment thereof.
The method of claim 1, wherein the CDR1 comprising the amino acid sequence of SEQ ID NO: 21, the CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and the CDR3 comprising the amino acid sequence of SEQ ID NO:
CDR1 comprising the amino acid sequence of SEQ ID NO: 27, CDR2 comprising the amino acid sequence of SEQ ID NO: 28 and CDR3 comprising the amino acid sequence of SEQ ID NO: 29,
CDR1 comprising the amino acid sequence of SEQ ID NO: 33, CDR2 comprising the amino acid sequence of SEQ ID NO: 34 and CDR3 comprising the amino acid sequence of SEQ ID NO: 35, or
An antibody comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 39, CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR3 comprising the amino acid sequence of SEQ ID NO: 41, or fragment thereof.
26. The method of claim 1, wherein the CDR1 comprising the amino acid sequence of SEQ ID NO: 24, the CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and the CDR3 comprising the amino acid sequence of SEQ ID NO: 26,
CDR1 comprising the amino acid sequence of SEQ ID NO: 30, CDR2 comprising the amino acid sequence of SEQ ID NO: 31 and CDR3 comprising the amino acid sequence of SEQ ID NO: 32,
CDR1 comprising the amino acid sequence of SEQ ID NO: 36, CDR2 comprising the amino acid sequence of SEQ ID NO: 37 and CDR3 comprising the amino acid sequence of SEQ ID NO: 38, or
An antibody comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 42, CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR3 comprising the amino acid sequence of SEQ ID NO: 44, or fragment thereof.
The method of claim 1, further comprising: a CDR1 comprising the amino acid sequence of SEQ ID NO: 21; a CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and a heavy chain variable region comprising CDR3 comprising the amino acid sequence of SEQ ID NO: A light chain variable region comprising CDR1 comprising the sequence, CDR2 comprising the amino acid sequence of SEQ ID NO: 25 and CDR3 comprising the amino acid sequence of SEQ ID NO: 26,
A heavy chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 27, CDR2 comprising the amino acid sequence of SEQ ID NO: 28 and CDR3 comprising the amino acid sequence of SEQ ID NO: 29, and CDR1 comprising the amino acid sequence of SEQ ID NO: A light chain variable region comprising CDR2 comprising the amino acid sequence of SEQ ID NO: 31 and CDR3 comprising the amino acid sequence of SEQ ID NO: 32,
CDR1 comprising the amino acid sequence of SEQ ID NO: 33, CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR3 comprising the amino acid sequence of SEQ ID NO: 35 and CDR1 comprising the amino acid sequence of SEQ ID NO: A light chain variable region comprising CDR2 comprising the amino acid sequence of SEQ ID NO: 37 and CDR3 comprising the amino acid sequence of SEQ ID NO: 38, and
A heavy chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 39, CDR2 comprising the amino acid sequence of SEQ ID NO: 40 and CDR3 comprising the amino acid sequence of SEQ ID NO: 41, and CDR1 comprising the amino acid sequence of SEQ ID NO: A light chain variable region comprising CDR2 comprising the amino acid sequence of SEQ ID NO: 43 and CDR3 comprising the amino acid sequence of SEQ ID NO: 44; or an antibody or fragment thereof comprising at least one heavy chain and light chain variable region selected from the group consisting of:
An amino acid sequence having 90% or more homology with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: / RTI &gt;
An amino acid sequence having 90% or more homology with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: / RTI &gt; And
An amino acid sequence having 90% or more homology with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: Lt; / RTI &gt; antibody or fragment thereof.
SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: An isolated anti-FcRn antibody or fragment thereof comprising at least one heavy chain and a light chain variable region.
7. An antibody or fragment thereof according to claim 6 comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO:
7. The antibody or fragment thereof according to claim 6, which comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO:
7. The antibody of claim 6, which comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12,
A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14,
A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16,
A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18, and
A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20; and a light chain variable region.
SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: A separate anti-FcRn antibody or fragment thereof comprising at least one heavy chain and a light chain variable region comprising an amino acid sequence that exhibits at least 90% homology.
11. The antibody or fragment thereof according to any one of claims 1 to 10, which binds to FcRn with a KD (dissociation constant) value of 0.01 to 2 nM at pH 6.0 and pH 7.4.
11. The antibody or fragment thereof according to any one of claims 1 to 10, which is a monoclonal antibody, a mouse antibody, a chimeric antibody, a humanized antibody or a human antibody.
11. The antibody of any one of claims 1 to 10, wherein the antibody is selected from the group consisting of a full-length antibody, Fab, F (ab ') ₂ , Fd, Fv, scFv, domain antibody, dual-specific antibody, bibodies, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient for the preparation of a medicament for the treatment and / or prevention of a disease, a body, a sterol regulatory binding protein cleavage activating protein, a bispecific antibody, a triple specific antibody, a multispecific antibody, diabody, triabody, tetrabodies, A domain immunoglobulin fusion protein, a camelized antibody, or an antibody comprising VHH, or a fragment thereof.
11. An antibody or fragment thereof according to any one of claims 1 to 10, comprising an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody.
17. A polynucleotide encoding an antibody of any one of claims 1 to 14 or a fragment thereof.
SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: RTI ID = 0.0 &gt; anti-FcRn &lt; / RTI &gt; antibody or fragment thereof.
SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: Lt; / RTI &gt; antibody or fragment thereof that exhibits at least 95% homology.
18. A recombinant expression vector comprising a polynucleotide according to any one of claims 15 to 17.
18. A host cell transfected with the recombinant expression vector of claim 18.
Culturing the host cell of claim 19 to produce an antibody; And
Separating and purifying the resulting antibody to recover an antibody that specifically binds to FcRn. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;
15. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 14 or a fragment thereof, and at least one pharmaceutically acceptable carrier.
21. A method of treating an autoimmune disease patient, comprising administering the composition of claim 21 to a patient.
24. The method of claim 22, wherein the autoimmune disease is selected from the group consisting of immunodeficiency neutropenia, gilanvalesindrome, epilepsy, autoimmune encephalitis, ischemic syndrome, birth syndrome, pemphigus pemphigus, decidual pemphigus, Idiopathic thrombocytopenic purpura, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, idiopathic thrombocytopenic purpura, acute myasthenia gravis, ITP), lupus nephritis, and membranous nephropathy.
15. An antibody or fragment thereof according to any one of claims 1 to 14 and a detection
A composition comprising a label.
15. A method for detecting FcRn in vitro or in vivo, comprising using an antibody according to any one of claims 1 to 14 or a fragment thereof.

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